DBC-DNA Adducts Research Articles

Transcriptional Profiling of Dibenzo[def,p]chrysene-induced Spleen Atrophy Provides Mechanistic Insights into its Immunotoxicity in MutaMouse.

Dibenzo[def,p]chrysene (DBC) is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) examined to date. We investigated the immunotoxicity of DBC, manifested as spleen atrophy, following acute exposure of adult MutaMouse males by oral gavage. Mice were exposed to 0, 2.0, 6.2, or 20.0 mg DBC /kg-bw per day, for 3 days. Genotoxic endpoints (DBC-DNA adducts and lacZ mutant frequency in spleen and bone marrow, and red blood cell micronucleus frequency) and global gene expression changes were measured. All of the genotoxicity measures increased in a dose-dependent manner in spleen and bone marrow. Gene expression analysis showed that DBC activates p53 signaling pathways related to cellular growth and proliferation, which was evident even at the low dose. Strikingly, the expression profiles of DBC exposed mouse spleens were highly inversely correlated with the expression profiles of the only published toxicogenomics dataset of enlarged mouse spleen. This analysis suggested a central role for Bnip3l, a pro-apoptotic protein involved in negative regulation of erythroid maturation. RT-PCR confirmed expression changes in several genes related to apoptosis, iron metabolism, and aryl hydrocarbon receptor signaling that are regulated in the opposite direction during spleen atrophy versus benzo[a]pyrene-mediated splenomegaly. In addition, benchmark dose modeling of toxicogenomics data yielded toxicity estimates that are very close to traditional toxicity endpoints. This work illustrates the power of toxicogenomics to reveal rich mechanistic information for immunotoxic compounds and its ability to provide information that is quantitatively similar to that derived from standard toxicity methods in health risk assessment.

Analysis of dibenzo[def,p]chrysene-deoxyadenosine adducts in wild-type and cytochrome P450 1b1 knockout mice using stable-isotope dilution UHPLC-MS/MS

The polycyclic aromatic hydrocarbon (PAH), dibenzo[def,p]chrysene (DBC; also known as dibenzo[a,l]pyrene), is a potent carcinogen in animal models and a class 2A human carcinogen. Recent investigations into DBC-mediated toxicity identified DBC as a potent immunosuppressive agent similar to the well-studied immunotoxicant 7,12-dimethylbenz[a]anthracene (DMBA). DBC, like DMBA, is bioactivated by cytochrome P450 (CYP) 1B1 and forms the reactive metabolite DBC-11,12-diol-13,14-epoxide (DBCDE). DBCDE is largely responsible for the genotoxicity associated with DBC exposure. The immunosuppressive properties of several PAHs are also linked to genotoxic mechanisms. Therefore, this study was designed to identify DBCDE-DNA adduct formation in the spleen and thymus of wild-type and cytochrome P450 1b1 (Cyp1b1) knockout (KO) mice using a highly sensitive stable-isotope dilution UHPLC-MS/MS method. Stable-isotope dilution UHPLC-MS/MS identified the major DBC adducts (±)-anti-cis-DBCDE-dA and (±)-anti-trans-DBCDE-dA in the lung, liver, and spleen of both WT and Cyp1b1 KO mice. However, adduct formation in the thymus was below the level of quantitation for our method. Additionally, adduct formation in Cyp1b1 KO mice was significantly reduced compared to wild-type (WT) mice receiving DBC via oral gavage. In conclusion, the current study identifies for the first time DBCDE-dA adducts in the spleen of mice supporting the link between genotoxicity and immunosuppression, in addition to supporting previous studies identifying Cyp1b1 as the primary CYP involved in DBC bioactivation to DBCDE. The high levels of DBC-DNA adducts identified in the spleen, along with the known high levels of Cyp1b1 expression in this organ, supports further investigation into DBC-mediated immunotoxicity.

Cancer Risk at Ultra-low Dose: Lessons Learned from 40,000-animal Cancer Dose-response Studies

There continues to be a shortage of ultra-low dose/response animal data on which to conduct modeling of human cancer risk. We have conducted two large-scale cancer and biomarker dose-response studies, one with dibenzo[def,p]chrysene (DBC, formerly called dibenzo[a,l]pyrene DBP) and a more recent study with aflatoxin B1 (AFB1), to address this need. These experiments used rainbow trout (Oncorhyncus mykiss), an animal model well suited to ultra low-dose carcinogenesis research, to explore dose-response down to a targeted 10 excess liver tumors per 10,000 animals (ED001). In study one, 40,800 trout were fed 0-225 ppm DBC for four weeks, sampled for biomarker analyses, and returned to control diet for nine months prior to gross and histologic examination. Suspect tumors were confirmed by pathology, and resulting incidences were modeled and compared to the default EPA LED10 linear extrapolation method. The study provided observed incidence data down to 2 above-background liver tumors per 10,000 animals at lowest dose (that is, an un-modeled ED0002 measurement). Among nine statistical models explored, three were determined to fit the liver data well—linear probit, quadratic logit, and Ryzin-Rai. None of these fitted models is compatible with the LED10 default assumption, and all fell increasingly below the default extrapolation with decreasing DBC dose. Low-dose tumor response was also not predictable from hepatic DBC-DNA adduct biomarkers, which accumulated as a power function of dose (adducts=100*DBC1.31). Two-order extrapolations below the modeled liver tumor data predicted DBC doses producing one excess cancer per million individuals (ED10−6) that were 500-1500-fold higher than that predicted by the five-order LED10 extrapolation. Study two was of similar design, but using AFB1. Analysis of the results is underway, and complicated by several differences from study 1, especially presence in some quartiles and treatment groups of a fatty liver syndrome. Preliminary logistic regression analysis excluding fish with this syndrome did not support the EPA linear default assumption (i.e., logistic slope 1.0), rather indicated a sublinear dose-response with slope of 1.42 (95% CI 1.23-1.61), and an extrapolated ED10−6 that is 32-fold greater than the LED10 default extrapolation. Inclusion of all fish also yielded a sublinear dose-response, with slope 1.31 (95%CI 1.13-1.50), and an extrapolated ED10−6 17-fold greater than the LED10 default extrapolation. Thus two genotoxins with differing biological properties yielded ultra-low dose-response curves in the same animal model that are statistically incompatible with the linear default assumption. These results are considered specific to the animal model, carcinogens, and protocols used. They provide the first experimental estimations in any model of the degree of conservatism that may exist for the EPA default linear assumption for a genotoxic carcinogen.

Cancer chemoprevention by dietary chlorophylls: A 12,000-animal dose–dose matrix biomarker and tumor study

Recent pilot studies found natural chlorophyll (Chl) to inhibit carcinogen uptake and tumorigenesis in rodent and fish models, and to alter uptake and biodistribution of trace 14C-aflatoxin B1 in human volunteers. The present study extends these promising findings, using a dose–dose matrix design to examine Chl-mediated effects on dibenzo(def,p)chrysene (DBC)-induced DNA adduct formation, tumor incidence, tumor multiplicity, and changes in gene regulation in the trout. The dose–dose matrix design employed an initial 12,360 rainbow trout, which were treated with 0–4000ppm dietary Chl along with 0–225ppm DBC for up to 4weeks. Dietary DBC was found to induce dose–responsive changes in gene expression that were abolished by Chl co-treatment, whereas Chl alone had no effect on the same genes. Chl co-treatment provided a dose–responsive reduction in total DBC-DNA adducts without altering relative adduct intensities along the chromatographic profile. In animals receiving DBC alone, liver tumor incidence (as logit) and tumor multiplicity were linear in DBC dose (as log) up to their maximum-effect dose, and declined thereafter. Chl co-treatment substantially inhibited incidence and multiplicity at DBC doses up to their maximum-effect dose. These results show that Chl concentrations encountered in Chl-rich green vegetables can provide substantial cancer chemoprotection, and suggest that they do so by reducing carcinogen bioavailability. However, at DBC doses above the optima, Chl co-treatments failed to inhibit tumor incidence and significantly enhanced multiplicity. This finding questions the human relevance of chemoprevention studies carried out at high carcinogen doses that are not proven to lie within a linear, or at least monotonic, endpoint dose–response range.

7H-dibenzo[c,g]carbazole metabolism by the mouse and human CYP1 family of enzymes

Found in tobacco smoke, fossil fuel and other organic combustion products, 7H-dibenzo[c,g]carbazole (DBC) is a potent mouse lung carcinogen and potential human carcinogen. Although the first hydroxylation is critical for determining activation versus detoxication, the enzymes responsible for site-specific hydroxylation of DBC are not known. We found that DBC-DNA adduct levels are significantly higher in aromatic hydrocarbon receptor null Ahr(-/-) mice, suggesting that the induction of Aromatic hydrocarbon receptor (AHR)-regulated genes, such as those in the CYP1 family, decrease DBC genotoxicity. Using knockout mice for Cyp1a1, Cyp1a2 and Cyp1b1, we showed that the major CYP1 enzymes that metabolize DBC are CYP1A1 in beta-naphthoflavone (BNF)-induced liver, CYP1A2 in non-induced liver, CYP1B1 and CYP1A1 in induced lung and none in non-induced lung. DBC metabolism by the human CYP1 enzymes was examined in vitro using Supersomestrade mark. Each mouse CYP1, as well as each human CYP1, has a unique DBC metabolite profile. Comparison of the metabolite profile in BNF-induced mice suggested that CYP1A1 primarily generates 1-OH, 2-OH and (5 + 6)-OH-DBC, whereas CYP1A2 generates primarily (5 + 6)-OH-DBC and CYP1B1 primarily generates 4-OH-DBC. This was similar to that observed in the human CYP1 enzymes. Most importantly, lung CYP1B1 is associated with forming 4-OH-DBC, the most potent metabolite leading to DBC-DNA adducts. These studies suggest that for non-pulmonary routes of exposure (i.e. skin, gastric, i.p.), low hepatic expression of CYP1A2 and CYP1A1, together with high expression levels of lung CYP1B1 and CYP1A1, may define a phenotype for high susceptibility to carcinogens such as DBC.

Impact of Cyp1a2 or Ahr gene knockout in mice: Implications for biomonitoring studies

Studies of the impact of phase 1 enzyme polymorphisms on genetic damage have yielded mixed results. We studied how genetic damage would be altered when specific genes were ablated under low dose conditions. Methods Knockouts (KO) were generated from c57bl6/J mice with mutations in Cyp1a2 or Ahr receptor that eliminated gene product function. Animals were treated topically with either 4-aminobiphenyl (4ABP) 10 mg/kg, benzo(a)pyrene (BaP) 33.3 mg/kg or dibenzo(c,g)carbazole (DBC) 8 mg/kg, and sacrificed after 24 h. DNA from livers, skin and/or urinary bladders were isolated and 32P-postlabelled. Results Cyp1a2−/− mice did not differ in 4ABP DNA adduct levels in either urinary bladder or liver compared to wildtype. There was a sex difference in the organ affected. Cyp1a2 knockout reduced skin BAP adduct levels 50% and AHR knockout reduced skin BAP adduct levels by 90%. There was no impact of either knockout on the levels of DBC-DNA adducts in any tissue. Conclusions Ablation of specific metabolizing enzymes had compound- and tissue-specific effects in mice. Phenotypic variability in single CYP enzymes may have minor impact in humans at low doses, but variation in the ability to induce the family of CYPs may have a greater impact.

In vitro toxicity of 7 H-dibenzo[ c,g]carbazole in human liver cell lines

7 H-Dibenzo[ c,g]carbazole (DBC) is a model N-heterocyclic aromatic compound (NHA) which is both a hepatotoxin and hepatocarcinogen in rodents. The focus of this investigation was to determine whether human liver cell lines display differential sensitivities to DBC-induced toxicity. Treatment of cell lines with increasing DBC concentrations produced apoptosis only in HepG2 cells. Although DBC inhibited the clonogenic growth of all cell lines at high concentrations, only the survival of HepG2 cells was reduced at lower concentrations. DBC inhibited DNA synthesis in two (HepG2, HLF) of the three cell lines at lower concentrations and was effective only at a high concentration in Mahlavu cells. Differences in DBC uptake were not observed in any of the cell lines, suggesting that bioavailability was not a limiting factor. DBC–DNA adducts were not detected in HLF or Mahlavu cells at either low or high concentrations of DBC. Consistent with the DNA adduct data, RP-HPLC analysis indicated that DBC was metabolized to a lesser degree in the HLF and Mahlavu cells. These results suggest that human liver cell lines differ markedly in the ability to metabolize DBC to toxic species and that DBC-induced apoptosis is only observed in cells that produce detectable metabolites and DBC–DNA adducts.

Comparative Oncogenic Activation of 7H-Dibenzo[C, G]Carbazole and Dibenz[A, J]Acridine

7H-Dibenzo[C, G]carbazole (DBC) is a potent liver, lung and skin carcinogen while dibenz[A, J]acridine (DBA) is a moderate skin carcinogen. DBC is metabolized to phenols and DBC-DNA adducts are formed through the 2-, 3-, and 4-phenols or directly through radical cations. Mutations in ras as a result of DBC, activation are found exclusively in codon 61 in liver, lung and skin. DBA is metabolized to dihydrodiols, and phenols and DBA-DNA adducts are formed through the diol-epoxides and possibly through bis-diol-epoxides. Mutations in ras, as a result of DBA activation, are found in codons 12, 13, and 61. These results indicate that although the two compounds are structurally similar, differing by one carbon in the middle ring, there are differences in their metabolism, DNA binding, mutational spectra and target-organ carcinogenesis.

One-electron oxidation is not a major route of metabolic activation and DNA binding for the carcinogen 7H-dibenzo[c,g]carbazole in vitro and in mouse liver and lung.

7H-Dibenzo[c,g]carbazole (DBC) is a potent multi-site, multi-species carcinogen present in a variety of complex mixtures derived from the incomplete combustion of organic matter. Like many carcinogens, DBC requires metabolic activation to an electrophilic species to exert its mutagenic and carcinogenic effects. One-electron oxidation, leading to the formation of radical cation intermediates, has been proposed as a mechanism of metabolic activation for DBC in vitro resulting in unstable DNA adducts. The purpose of this research was to determine whether one-electron oxidation is a mechanism of activation and DNA binding for DBC in vivo. Specific depurinating DBC-DNA adducts formed by one-electron oxidation were analyzed in mouse liver at 4 h following a single i.p. dose of 40 mg/kg of 11 microCi [(14)C]DBC. In addition to five previously published adduct standards, two newly identified adduct standards were characterized by mass spectrometry and NMR, namely DBC-6-N7-Ade and DBC-6-N1-Ade; however, neither was observed in mouse liver. Only the DBC-5-N7-Gua adduct was observed in mouse liver extracts at a level of 6.5 +/- 1. 8 adducts/10(6) nucleotides. In addition, the formation of AP sites and stable DBC-DNA adducts was analyzed in mouse liver and lung at 4, 12 and 24 h following a single i.p. dose of 0.4, 4 or 40 mg/kg DBC (n = 3/group). There was a distinct time- and dose-response of stable DBC-DNA adducts detected by (32)P-post-labeling. There was not a clear dose-response for formation of AP sites; however, a significant increase over control levels was observed at the 4 and 40 mg/kg dose groups at 4 and 12 h post dosing, respectively. Quantitative comparisons indicate that the depurinating DBC-5-N7-Gua adduct constitutes approximately 0.4% of total adducts measured whereas the stable adducts detected by (32)P-post-labeling constitute 99.6% of total adducts measured following a 40 mg/kg dose and a 4 h time-point. The data indicate that one-electron oxidation does occur in mouse liver in vivo. However, one-electron oxidation is a minor mechanism of activation in that the percentage of total adducts formed through this route constitutes a minor percentage of the total adducts formed.

A Comparison of Apoptosis and Necrosis Induced by Hepatotoxins in HepG2 Cells

7H-Dibenzo[c,g]carbazole (DBC), an N-heterocyclic aromatic hydrocarbon, is cytotoxic and carcinogenic in rodent liver. While DBC leads to necrotic lesions in the liver, the induction of apoptosis by DBC has not been investigated. The focus of this study was to determine the degree to which apoptosis and necrosis contributed to DBC cytotoxicity in a human hepatoma cell line (HepG2). To determine if these effects were unique to DBC, the results were compared to another hepatotoxin, aflatoxin B1 (AFB1). DBC produced a distinct biphasic LDH release curve within 24 h of exposure. During the same time period lower concentrations of DBC (<10 μM) induced the formation of DBC–DNA adducts and increased p53 protein levels followed by apoptotic cell death. However, increasing the concentration of DBC to 80 μM led to lower DNA adduct and p53 protein levels. At this concentration, intracellular ATP levels were rapidly depleted followed by cell swelling and loss of membrane integrity consistent with necrotic cell death. In contrast to DBC, a biphasic LDH release curve was not observed for AFB1. Instead, AFB1 induced a concentration-dependent increase in apoptosis that reached two- to threefold higher levels than DBC. These results suggest that differences exist in the extent and type of cell death induced by DBC and AFB1 at equimolar concentrations. Apoptosis and necrosis result from low and high concentrations of DBC, respectively, and may be dependent upon intracellular ATP levels.

Combination of high-performance liquid chromatography and thin-layer chromatography separation of five adducted nucleotides isolated from liver resulting from intraperitoneal administration with 7H-dibenzo[ c, g]carbazole to mice

7H-Dibenzo[ c, g]carbazole, DBC, is a potent environmental liver carcinogen. Liver DNA from mice treated with DBC exhibited seven distinct DBC-DNA adducts as detected by 32P-postlabeling using multidimensional TLC. To improve quantitation and chemically characterize the adducts, DNA samples were hydrlyzed, 32P-postlabeled and the adducts were separated from the unadducted normal nucleotides on TLC using a D1 solvent, 0.65 M sodium phosphate (pH 6.8). Adducts were eluted from the TLC plates with 4.0 M pyridinium formate, concentrated, resuspended in 50% aqueous methanol and injected onto the HPLC; five individual adduct peaks were resolved and collected by this method. This approach will prove useful to decrease analysis time and improve chemical characterization of tightly clustered DNA adducts generated in vivo.

7H-Dibenzo(c,g)carbazole-DNA Adducts in Target and Non-target Organs of Mice Exposed by Acute and Chronic Administration

Abstract Carcinogen-DNA adducts appear to be biomarkers of effective dose and/or early effect. The relationship between these biomarker levels in target and non-target organs under acute and chronic exposures is important. 7H-Dibenzo(c,g)carbazole(DBC) has been shown to induce tumors in experimental animals. The time courses of DBC-DNA adduct formation in skin, liver and lung following both single (i.p.) and repeated (topical) administration of DBC were investigated. In the acute study, DNA binding levels increased rapidly in the lung over 3–7 days at all doses and then decreased slowly over 21 days; levels in the liver were 10 times that in the lung with similar increases in levels over time. In the chronic study, levels increased with time over 15 weeks with the liver binding levels 8–10 times that seen in skin and lung. In both studies the major adduct in liver was adduct 6, and in the lung adduct 3, while in skin adducts 2 and 3 were the major adducts. These data indicate that the adduct pattern is ta...

Carcinogenicity, DNA adduct formation and K-ras activation by 7H-dibenzo[c,g]carbazole in strain A/J mouse lung.

N-Heterocyclic polynuclear aromatic hydrocarbons (NHA) are environmental pollutants formed during the combustion of organic materials. 7-H-Dibenzo[c,g]carbazole (DBC) is a potent carcinogen in lung, liver and skin. We undertook these studies to determine whether tissue specificity for DBC lung carcinogenicity in the strain A/J mouse is mirrored by formation of DBC-DNA adducts in lung tissue and whether these adducts are consistent with mutation patterns in the K-ras gene. Strain A/J mice were given a single i.p. injection of DBC at doses of 0, 5, 10, 20 or 40 mg/kg and levels of DNA adducts in the lung were monitored by 32P-postlabeling on days 1, 3, 5, 7, 14 and 21. The remaining animals were sacrificed 8 months after DBC treatment and lung tumor multiplicity and K-ras mutation patterns in the tumors were determined. The lung tumor response to DBC was dose related, with an average of 4.7 +/- 1.2 tumors/mouse at 5 mg/kg and 48.1 +/- 5.5 tumors/mouse at 40 mg/kg. As many as seven DBC-DNA adducts were observed in the lung. DNA binding levels in the lung were highest at 40 mg/kg, with maximum binding at 5-7 days. At lower dose levels the maximum binding to DNA decreased and shifted to earlier time points. The DBC-DNA adduct in the lung with the highest level of binding at all dose levels was DBC-DNA adduct 3. The majority of DBC-induced mutations in the K-ras gene in the lung were A-->T (80%) transversions in the third base of codon 61, a mutation that has not been previously observed in chemically induced lung tumors in strain A/J mice.

Target Organ Specificity For N-Heterocyclic Aromatics

Abstract N-Heterocyclic aromatics are environmental carcinogenic pollutants. Of these compounds, 7H-dibenzo[c,g]carbazole (DBC) is a liver and skin carcinogen in the mouse by topical application and a lung carcinogen by IP injection, whereas dibenz[a,j]acridine (DBA) is a skin carcinogen by topical application. Using 32P-postlabeling techniques, it was found that DBC-DNA adduct 6 is the major adduct in the liver, consistent with the metabolism of DBC to the 3-OH-DBC; adducts 2 and 3 are major adducts in the skin and adduct 3 is the major adduct in the lung. On the other hand, DBA adduction is apparent only in the skin and is consistent with the initial metabolism to DBA-3, 4-dihydrodiol. These data indicate that the adduct pattern, as well as the proximate metabolites, are target organ specific; these data will have an impact on the development of biomarkers of the carcinogenic process.

Tissue distribution of DNA adducts of 7H-dibenzo[c,g]carbazole and its derivatives in mice following topical application.

7H-Dibenzo[c,g]carbazole (DBC) is a potent liver and skin carcinogen following topical administration. The objective was to determine the pattern of DBC-DNA adducts produced in both target and nontarget tissues when DBC and its metabolites were applied topically at carcinogenic doses. DBC phenolic derivatives 1-hydroxy-DBC, 2-hydroxy-DBC, 3-hydroxy-DBC, 4-hydroxy-DBC, 5-hydroxy-DBC, 6-hydroxy-DBC, 13-c-hydroxy-DBC, and N-methyl-DBC were applied dermally to Hsd:ICR (Br) mice. Tissues were harvested 24 h later, and DBC-DNA adduct levels were determined by 32P-postlabeling. The levels of DBC-DNA adducts were about 25 times greater in liver than in any other tissue. Total DBC-DNA adducts were seen in skin and lung at about equal levels, while adduct levels in kidney and other tissues were no more than one fourth that of lung and skin. Adduct 6 was the predominant adduct in liver, adducts 2 and 3 were formed preferentially in skin, and adduct 3 was formed preferentially in lung. 3-Hydroxy-DBC and 4-hydroxy-DBC produced higher levels of DNA adducts in skin, lung, and liver than did the parent compound or 2-hydroxy-DBC. DNA adducts were not seen in any tissue for the 1-, 5-, 6-, or 13-c-hydroxy compounds. In addition, hepatic DNA adducts were not seen when the nitrogen of DBC was methylated. In lung and skin, N-methyl-DBC induced DNA adducts at levels comparable to DBC, although the adduct profile in these tissues was different from that of DBC itself.(ABSTRACT TRUNCATED AT 250 WORDS)

32P-Postlabeling analysis of DNA adduct formation and persistence in English sole ( Pleuronectes vetulus) exposed to benzo[ a]pyrene and 7H-dibenzo[c,g]carbazole

The formation and persistence of benzo[ a]pyrene (BaP)- and 7H-dibenzo[c,g]carbazole (DBC)-DNA adducts in liver of English sole ( Pleuronectes vetulus) were investigated. BaP is a putative hepatocarcinogen in English sole based on its ability to induce formation of preneoplastic foci, while DBC is a hepatocarcinogen in mammals but whose carcinogenicity in fish is not known. English sole liver was sampled from 2 h through 84 days after a single intermuscular injection of a BaP and DBC mixture (100 μmol of each/kg body wt.), and DNA adduct levels were measured by the nuclease P1 version of the 32P-postlabeling assay. The major BaP adducts detected were from binding of BaP-7,8-diol-9,10-epoxide to DNA, whereas multiple uncharacterized DBC-DNA adducts were detected. Total adduct levels for both BaP and DBC reached a maximum at 2 days post exposure. The levels of DBC-DNA adducts were greater than the levels of BaP adducts at all time points and increased more rapidly than did the levels of BaP-DNA adducts. The DBC to BaP adduct ratio was 33 ± 8.8 at 2 h and declined to 4.2 ± 0.48 by 12 h post exposure. From 2 to 28 days, the levels of both BaP and DBC adducts declined with apparent half-lives of 11 and 13 days, respectively. There was no apparent decline from 28 to 84 days in the levels of the remaining BaP or DBC adducts; these persistent adducts represented 32 and 36% of maximum levels, respectively. These results provide the first data on the kinetics of adduct formation and removal of a carcinogenic nitrogen-containing polycyclic aromatic compound in fish. The results showing greater binding and similar persistence of DBC-DNA adducts compared to BaP-DNA adducts suggest that DBC may be hepatotoxic and potentially carcinogenic in English sole. In a separate experiment, the effect of multiple doses of BaP (30 μmol/kg body wt.) on the levels of hepatic BaP-DNA adducts showed that adduct levels increased linearly ( r = 0.815, P = 0.0007) with 5 successive doses administered at 2 day-intervals and sampled 2 days after the last dose. The persistence of both BaP-DNA and DBC-DNA adducts in liver, together with the increase in BaP-DNA adducts in English sole exposed to successive doses of BaP, suggest that hepatic xenobiotic-DNA adducts in English sole are molecular dosimeters of relatively longterm environmental exposure to genotoxic polycyclic aromatic compounds.

32P-postlabeling analysis of DNA adduction in mice by synthetic metabolites of the environmental carcinogen, 7H-dibenzo[c,g]carbazole: chromatographic evidence for 3-hydroxy-7H-dibenzo[c,g]carbazole being a proximate genotoxicant in liver but not skin.

The DNA adduction by the environmental carcinogen 7H-dibenzo[c,g]carbazole (DBC) and chemically synthesized 2-OH, 3-OH, and 4-OH metabolites of DBC was investigated in liver and skin of female CD-1 mice. After topical application to the skin of 37 mumol/kg of DBC or the phenolic metabolites, DNA adducts were measured by a 32P-post-labeling assay employing carrier-free [gamma-32P]ATP and ATP-deficient conditions. In liver, DBC produced four major and several minor chromatographically distinct adducts of as yet undetermined chemical structure. The adduct pattern elicited by 3-OH-DBC was qualitatively similar to the DBC adduct pattern, while this was not the case for 2-OH-DBC and 4-OH-DBC. On the basis of co-chromatography experiments under various conditions, the DBC and 3-OH-DBC adducts appeared identical, and the total of adduction elicited by these compounds in liver was substantial. Similar results were observed when DBC or 3-OH-DBC were administered i.p. As a major difference between the two compounds, one 3-OH-DBC adduct (no. 3) was 4.4- and 7.0-fold lower than the corresponding DBC adduct after i.p. and topical dosing, respectively. In skin, DBC produced two major adduct fractions after topical application, one of which could be chromatographically resolved into three subcomponents. Prominent adducts produced in skin DNA by each of the three metabolites were different from those elicited by DBC, and the level of adduction by the metabolites was significantly lower than that by DBC. Comparison of the skin and liver DBC-DNA adduct patterns after topical application of DBC showed that only one of the four major chromatographically resolved skin adducts corresponded to a major liver adduct (no. 3), and that total adduction in liver was 13.5-fold higher than in skin. These results suggested that activation of DBC to DNA-binding compounds in liver occurs through at least two pathways with 3-OH-DBC being a proximate carcinogen involved in the formation of most of the adducts; 3-OH-DBC and the other two phenolic metabolites investigated play a minor role, if any, in the formation of DBC-DNA adducts in skin; metabolic activation of DBC to DNA-binding compounds in liver and skin appears to follow pathways that are different in terms of both the chemical nature and the amount of the adducts formed; and DBC and 3-OH-DBC exhibit a strong preference for liver versus skin DNA.